We are continuing to develop an in vitro model to check anticataract agents. The organ culture system utilizes lenses from rats and monkeys. We have developed a method to screen the lenses to determine which might have been damaged in dissection. This is the first method to adequately predict the integrity of the lens at a very early stage in culture; lens clarity previously had been taken as a measure of integrity. Clarity at a very early stage in the culture process has not always been correct. Many lenses can maintain clarity but are not able to transport ions and amino acids normally. By analyzing the culture medium, we also have been able to show that the main protein in the organ culture medium is albumin, most probably as a residual protein sticking to the lens during dissection. The albumin can be taken out of the medium, and with a viable lens, very little protein will leak out of the lens. Curiously, when the lens is challenged with hydrogen peroxide as a model for oxidative stress, the beta B-2 crystallin is one of the principal proteins lost by the lens. Additional studies of the organ-cultured lenses have begun analysis of the types of messenger RNA expressed in the lens under stress. We are using monkey lenses to determine the sequences of these stress-related messages. Work on the lens epithelium has continued to study two specific questions: One concerns the protective mechanisms present in the lens epithelium to prevent damage from oxidative stress. Work with the lens epithelium cell lines has shown that the major oxidoreductase, is responsive to the oxidative stress system appears to be D-T diaphorase. Zeta-crystallin, also an oxidoreductase activated in an oxidative stress and increase in the lens cells. The increase in the zeta-crystallin cannot by itself account for the large increase in oxidoreductase found in the stressed cells. The second question concerns cellular differentiation into fiber cells. The region of the lens where this process occurs tends to be the one most highly altered under stress conditions. We have separated certain steps in the differentiation process and will be better able to explore the stages at which cataracts might develop in the equatorial region of the lens. Work continues on the human beta B-2 crystallin, which now has been successfully cloned and sequenced. The deduced sequence recently has been confirmed by another lab. Since this protein is developmentally regulated, investigation into the promoter activity of this gene is continuing.